The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extra-cellular matrix (ECM) has been a major objective for the tissue engineering field. Atomic force microscopy verified the fibrilar structure of the gels and the mechanical properties were characterized for various weight XR9576 percent (wt%) formulations of the 5 mol% PR_g – 95 mol% E2 peptide-amphiphile mixture. The 0.5 wt% formulations had an elastic modulus of 429.0 21.3 Pa while the 1.0 wt% peptide-amphiphile hydrogels had an elastic modulus of 808.6 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus and the decrease was a function of the cell loading. While both formulations supported cell proliferation, the 0.5 wt% gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt% gels. However, compared to the 0.5 wt% formulations, the 1.0 wt% hydrogels promoted greater increase in mRNA expression and production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advance of 3D culture applications and cell therapies. INTRODUCTION The importance of the mechanical, structural, and biochemical properties of the native ECM has been recognized and has led to the development of new techniques and chemistries to form and functionalize scaffolds for cell culture. In addition to designing scaffolds which mimic the nanofibrous structure, stiffness and biochemical signals of the ECM, mimicking the innate 3D environment of the ECM have been shown to be critically important for tissue engineering applications.1C4 Traditional 2D cell culture introduces an artificial polarity between cells upper and lower surfaces resulting in non-native cell morphologies and receptor and protein expression profiles. 5 Additionally, significant differences in the expression of integrins and other cell surface receptors between 2D and 3D environments have been reported.6,7 Encapsulation of cells within a 3D scaffold is most effectively accomplished by introducing cells during gelation in order to ensure uniform cell distribution. Many of the common hydrogel systems currently used for 3D cell entrapment require low pH, as with PuraMatrix;8 low temperature, XR9576 as with collagen and Matrigel;9 or HRY exposure to UV light to initiate XR9576 gelation, as with polyethylene glycol (PEG),10,11 which can damage cells and lead to cell death. Peptide-amphiphiles are an attractive material for the design of cell scaffolds which mimic the ECM due to their ability to self-assemble into nanofibrous hydrogels and incorporate relevant bioactive, biomimetic peptide sequences.12 Peptide-amphiphiles have been shown to form hydrogels in cell culture media and have been used as 3D scaffolds for a variety of applications, including the culture and differentiation of dental stem cells,13,14 mesenchymal stem cells,15C17 neural progenitor cells,18C20 cartilage, 21 and pancreatic islets.22,23 It has been demonstrated that RGD-containing peptide-amphiphile scaffolds support enhanced proliferation and osteogenic differentiation of mesenchymal stem cells compared to peptide-amphiphile scaffolds without the RGD peptide.16 Peptide-amphiphiles containing the laminin mimetic peptide, IKVAV, have been shown to support neuron differentiation of neural progenitor cells.18 Peptide-amphiphiles gels have also been shown to be biocompatible in vivo, with significant degradation within the first 30 days of XR9576 implantation, followed by complete degradation after 60 days with no signs of acute or chronic inflammation.24 Other work has shown that entrapped cells internalize the peptide- amphiphile nanofibers and possibly utilize peptide-amphiphiles in their metabolic pathways.25 We have previously developed a fibronectin-mimetic peptide-amphiphile, called PR_g, which self-assembles in water to form nanofibrous hydrogels.26 The PR_g peptide contains both the primary cell binding site, RGD, as well as the PHSRN synergy site. These two sites are separated by a linker which accurately mimics the distance and the overall hydrophobicity/hydrophilicity between these binding domains in fibronectin.27,28 We have previously demonstrated that the PR_g peptide specifically binds the 51 integrin27 with a dissociation constant of 76.3 6.3 nM.29 PR_g peptide-amphiphile hydrogels have been shown to support increased cell adhesion, proliferation, and ECM secretion as 2D.